In recent years, from the viewpoint of environmental problems such as global warming and air pollution, the development of new energy as an alternative to fossil fuel, such as photovoltaic power generation, wind power generation, and geothermal power generation is being advanced. In particular, as a means of suppressing carbon dioxide emissions, the use of plant biomass as renewable energy has been attracting attention. Plant biomass is mainly composed of cellulose, hemicellulose and lignin. Biological methods, physical methods and chemical methods are available as methods of hydrolyzing plant biomass, and biological hydrolysis methods by enzymes (cellulases) are the current mainstream. Cellulose and hemicellulose are hydrolyzed to form monosaccharides such as glucose and xylose, which can then be used as biofuels or the raw materials for chemical products.
Lignocellulose is recalcitrant due to its highly complex structure, and is difficult to degrade or hydrolyze with a single cellulolytic enzyme. Accordingly, among the various polysaccharides, hydrolysis of cellulose generally requires three types of glycoside hydrolase enzymes, namely an endoglucanase (endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21). On the other hand, hydrolysis of hemicellulose requires a xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and a β-xylosidase (3.2.1.37).
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the hydrolyzed biomass solution is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis process at a high temperature, for example 80° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the hydrolyzed biomass solution can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermal stability is very desirable.
For the purpose of lignocellulose degradation, use as a processing agent of cellulose fibers, and pulp and paper processing, numerous thermostable enzymes that can be used for lignocellulose hydrolysis treatment at a high temperature, especially endoglucanases necessary for the hydrolysis of cellulose, have been isolated to date from thermophilic bacteria, filamentous fungi, archaea and the like (for example, see Patent Document 1). In addition, attempts have also been made to further improve the specific activity and the thermal stability, for example, by using the mutants of a host organism or partially modifying the amino acid sequences of these enzymes. However, almost all of the above enzymes have optimum temperatures of 60 to 80° C., and further improvements in the thermal stability are still required. On the other hand, there are hyperthermostable endoglucanases having an optimum temperature of more than 90° C. For example, in Patent Document 2, Rhodothermus marinus has been reported to have an endoglucanase Cel12A with an optimum temperature of more than 105° C.